Project Summary In many biological systems G protein-coupled receptors (GPCRs) provide a crucial molecular link between the dynamics of the extracellular environment and the associated intracellular signaling response. In the nervous system, GPCRs serve as detectors of precise patterns of neurotransmitter release and are able to, in turn, modulate neuronal excitability and synaptic transmission. Of particular importance are the class C metabotropic glutamate (mGluR) and GABA receptors (GABABR), which respond to the major excitatory and inhibitory neurotransmitters, respectively, and serve as drug targets for neurological and psychiatric disorders. Unfortunately, our understanding of their underlying molecular mechanisms of signaling remain limited due to a lack of methods for the direct measurement and manipulation of their activity with high specificity and spatial and temporal precision. Furthermore, the biophysical activation mechanism of class C GPCRs is particularly challenging to decipher because unlike class A GPCRs, such as rhodopsin or -adrenergic receptors, they contain large, extracellular ligand binding domains (LBDs) that multimerize and couple, via a poorly understood mechanism, to a transmembrane domain (TMD). Our recent work has established new optical methods for directly measuring mGluR assembly and conformational dynamics at the single molecule level and has also produced an optogenetic method to manipulate receptors with subtype selectivity and high spatiotemporal precision using photoswitchable tethered ligands. These breakthroughs have advanced our understanding of how mGluRs dimerize and the initial molecular motions that lead to cooperative receptor activation, but many fundamental questions remain. In research area 1 we will dissect the activation mechanism of mGluRs and GABABRs in a quantitative, interdisciplinary way using optical approaches, including single molecule Forster resonance energy transfer (FRET) to measure conformational dynamics, in conjunction with functional reporters and detailed structural analysis. The long-term goal is to understand, biophysically, how allosteric inter-domain and inter-subunit coupling interactions permit orthosteric and allosteric ligand binding to produce G protein activation. This work will give major insight into the fundamental activation processes of a large class of membrane receptors and should provide a deeper understanding of their molecular pharmacology. In research area 2 we will improve and harness the power of optical sensors of activation and optogenetic control of receptors to probe the kinetics of different mGluR subtypes at the level of activation, signaling, and desensitization and to dissect their spatiotemporal signaling profiles at hippocampal synapses. In the long term we plan to use this information to probe the mechanism of induction of long-term depression by pre-synaptic, post-synaptic, and glial mGluR populations. This work will provide a dynamic picture of mGluR signaling that has been missing from the field and will strengthen our molecular understanding of the role of these receptors in synaptic modulation in health and disease.